Pancreatic cancer (PaC) ranks the fourth in the US and eighth worldwide for cancer mortality. Early diagnosis and prevention is the only viable option for impacting the mortality rate. Due to the low prevalence of PaC in the population, however, most positive screening tests are likely false positives. Therefore, it remains a grand challenge to identify more accurate biomarkers for early screening and prognosis prediction. Given the complex pathogenesis of PaC, identification of biomarkers for PaC will benefit from a systematical search targeting various gene regulation mechanisms, considering that gene expression dysregulation underlies the disease development. Recent studies using high-throughput epigenomic profiling technologies have demonstrated aberrant modifications of CpG sites in PaC cell lines and tumor tissues, suggesting the potential applications of these epigenetic markers to early detection of this dreadful disease. In particular, 5- hydroxymethylcytosine (5-hmC), which can be generated by oxidation of 5-methylcytosine (5-mC) through the Tet family of enzymes, was recently re-discovered to be a stable base in various human tissues, with emerging gene regulatory role and implications for human cancers. We reasoned that determining the exact types of cytosine modifications (5-mC or 5-hmC) implicated in PaC will provide novel insights into its pathogenesis, as well as novel epigenetic biomarkers for PaC early detection. Specifically, The Cancer Genome Atlas (TCGA) data will be explored to guide the selection of PaC-specific CpGs to be tested for the existence of 5-hmC in a panel of PaC cell lines with varying degrees of genetic complexity using a novel and proven technique, Tet- assisted Bisulfite Sequencing (TAB-Seq), developed by our colleague and collaborator, Dr. Chuan He (HHMI Investigator). We will also use the TAB-Seq assay to examine 5-hmC directly in PaC tumor and surrounding normal tissue samples that have already been collected from 30 US and 30 Chinese patients in our previous or ongoing projects. Our design of including two diverse populations will allow investigation of the role of these epigeneti markers in the health disparities in this disease. To examine the clinical significance of the 5-hmC, we will evaluate our detected CpG markers (5-mC and 5-hmC) in cells from endoscopically- and histologically normal-appearing periampullary duodenal biopsies from 30 US and 30 Chinese PaC patients as well as 30/30 matched controls in relation to the nanoscale cellular architecture alterations due to field carcinogenesis using a novel biophotonic technology. In summary, this project will for the first time define which cytosine modifications, 5 hmC or 5-mC or both, are implicated in PaC. The results from this proposal will lay the foundation of applying CpG markers including 5-hmC as a novel class of early screening and predictive biomarkers for PaC. Finally, the PIs, Drs. Zhang and Hou have assembled an excellent investigative team with significant achievements in relevant research areas and complimentary expertise, therefore ensuring the success of this multidisciplinary and highly innovative project.